The vertebrate immune system is characterized by its ability to respond to an enormously diverse set of antigenic determinants. This capability is due to the synthesis by the body of a set of glycoproteins whose specificity for a single antigen is determined by a variable sequence of amino acids which binds to the antigen. The glycopcoteins, which recognize and bind free antigens, are produced by B cells and are called immunoglobulins (Ig).
Each B cell, or bone marrow-derived lymphocyte, produces antibody specific for only one antigen. It has been theorized that the type of immunoglobulin which is produced by the B cell is generated by a series of gene rearrangements and RNA splicing events that result in polypeptide chains consisting of variable and constant regions. These regions can be subdivided into domains held together by interchain and intrachain disulfide bridges situated at the same relative positions. The characteristic primary and secondary structure is made up of heavy and light chains which begin with a leader peptide of 17-29 residues, followed by a variable (V) region of 94-97 residues, then a joining region of 13-17 residues (J), then a constant region (C). The domains of the constant regions of the immunoglobulins are encoded by separate exons from those for the variable region and do not appear to rearrange during development.
T cells or thymus derived lymphocytes, like B cells, are capable of recognizing a wide range of different antigens. The ability to recognize a given antigen is also fixed in any particular clonal line of T cell. T cells, however, recognize only antigens located on the surfaces of cells in the specific, molecular context of self major histocompatability complex (MHC) gene products, not freely circulating antigens. T cell receptors recognize foreign antigens (such as viral antigens) in the molecular context of the T cell host's self-MHC gene products or recognize foreign MHC gene products. Cell surface antigens include tumor cell and viral antigens. The ability to recognize cell-bound antigens is acquired when the T cells differentiate in the host thymus.
Effective antisera and monoclonal antibodies have now been developed which recognize and precipitate clone-specific proteins on the surface of functional T cell clones, hybridomas or T cell tumors. Studies using these antibodies have suggested that the specificity--determining portion of a T cell receptor is a heterodimeric glycoprotein of about 90,000 daltons and consisting of an alpha subunit and a beta subunit held together by an inter-chain disulphide bond(s). Peptide fingerprint analysis of alpha and beta chains from several T-cell lines suggest that both subunits are composed of variable (V) and constant (C) regions.
Three groups of workers have since succeeded in isolating T cell-specific cDNA (complementary DNA) clones of mouse or human origin which act homologous to immunoglobulin genes. S. M. Hedrick, E. A. Nielsen, J. Kevaler, D. I. Cohen, and M. M. Davis, as reported in Nature, 308, 153-158 (1984), using a mouse, antigen specific, MHC-restricted T helper hybridoma TM86, demonstrated that the cDNA encded a pcotein composed of an amino-terminal variable and a carboxy-terminal constant region. They also showed that the corresponding genomic DNA sequences had undergone clone-specific somatic rearrangements in various T cell lines.
Y. Yanagi, Y. Yoshikai, K. Leggett, S. P. Clark, I. Aleksander, and T. W. Mak, in Nature, 308, 145-149 (1984), reported the nucleotide sequence of a cDNA clone YT35 derived from the human leukaemic T cell line, MOLT-3. The predicted amino acid sequence encoded bay the human cDNA clone of Yanagi et al. is highly homologous in the constant region to the cDNA clone isolated by Hedrick et al.
We recently reported in Nature, 309, 757-762 (1984) the cloning and sequencing of two related but distinct cDNA clones pHDS4/203 and pHDS11 from a functional murine cytolytic T lymphocyte clone, 2C, the teachings of which are incorporated herein. The genes corresponding to these cDNA clones are rearranged and expressed in T cells (and not in other cells that have been examined such as B cells, kidney cells, etc.) and also have significant sequence homologies to immunoglobulin V and C genes. The amino acid sequence encoded by clone pHDS11 corresponds to the same T-cell receptor genes as those isolated by Hedrick et al and Yanagi et al.
A second cDNA clone pHDS4/203 was identified as the gene coding for the alpha chain of the T cell antigen ceceptor. However, subsequent studies have shown that this clone lacks sequences that correspond to sites for N-linked glycosylation and it has recently been shown that both the alpha and beta chains of the T cell receptor are N-glycosylated. Despite the fact that the four clones: YT35, isolated by Yanagi et al, TM86, isolated by Hedrick et al, and pHDS11 and pHDS4/203, isolated by Saito et al, share several common properties, it is unlikely that the clone pHDS4/203 encodes for the alpha subunit of the T cell receptor protein comprising the beta subunit encoded by the genes YT35, TM86, and pHDS11. These four cDNA clones share the following common properties. They are expressed in T cells but not in B cells. The corresponding genes are rearranged in T cells and not in B cells. In the encoded proteins, there are distinctive regions that, proceeding from amino- to carboxy-terminus, correspond to a signal peptide, two immunoglobulin-like domains, a transmembrane peptide rich in hydrophobic amino acids, and a short cytoplasmic peptide. Their deduced amino acid sequences have low but significant homology to those of immunoglobulin-chains. Each gene is composed of separate V, J and C gene segments. In the cases of YT35 and TM86, the corresponding genes have also been shown to have D segments. Their V, (D), and J segments have characteristic signal sequences foc gene rearrangement (heptamer and nonamer separated by either 12 or 23 base pairs).
Due to the substantial homology between the sequences of TM86, YT35 and pHDS11, it is likely that they represent the beta subunit of the T cell receptor.
Due to the absence of N-glycosylation sites on the gene PHDS4/203 Saito et al, it is unlikely that this gene represents the alpha subunit of the T-cell receptor.
It is therefore an object of the present invention to provide a T-cell receptor gene or nucleotide sequence which codes for both subunits of the T-cell receptor.
It is a further object of the present invention to provide murine cDNA clones which code for either the alpha or beta subunit of the T cell receptor.
It is a still further object of the present invention to provide hybridization probes for identifying and isolating the T cell receptor genes and subunits of the T cell receptor genes of other, non-mouse species, including the human specie.
Another object of the present invention is to provide the protein or amino acid sequence of a murine alloreactive cytotoxic T lymphocyte receptor, including both of its subunits.
It is yet another object of the present invention to provide specific antibodies to the T cell receptor and subunits of the T cell receptor which are useful for identification, isolation, and in other methods for which antibodies are useful such as in delivering antibody bound drugs to a specific cell.